Summary: The immune response to polysaccharide (PS) antigens is highly regulated and has several distinguishing features including restricted subclass, variable region gene usage, fine specificity, and avidity. Simple PS not conjugated to protein (such as bacterial Levan (BL, Neisseria meningitidis group C (MCPS)) elicit a thymus-independent (TI) response. PS conjugated to proteins (such as MCPS coupled to tetanus toxoid, (MCPS-TT)), on the other hand, elicit a different type of response, termed thymus-dependent (TD). Our earlier analysis of anti-BL antibodies had shown a germline primary response to the inulin (In) branch determinants. Two injections of BL, however, elicited IgM mAb with somatic mutations and higher affinity. We have now performed site-directed mutagenesis of the germline antibodies and identified specific sites resulting in higher or lower affinity. Data show the importance of aromatic and basic amino acids in the CDRs of anti-PS antibodies. In our analysis of the regulation of diversity in the anti-In response we have mapped the Sr1 diversity gene to mouse chromosome 14. Recent studies in TCR KO mice have shown that the response to In is not only TI, but is not influenced by T cells when added back, despite a large increase in serum IgG and IgM. Previous analyses of anti-MCPS and anti-MCPS TT mAb reveal that VH gene family usage is dominated by VHJ558. Sequence analysis of anti MCPS mAb shaows that several different germline genes are used in this response, even by mAb of the same fine specificity. Few somatic mutations are seen in response to MCPS, but when observed, correlate with an increase in affinity. Recent modeling studies suggest that the presence of basic amino acids in the combining site aslo correlate with an increase in affinity. Earlier mouse model studies in our laboratory indicated a developmental delay in the immune response to MCPS even when it was administered as TD MCPS-TT conjugates. As part of these studies, T cell clones were generated from mice immunised with MCPS TT. These clones have been shown to include specificities for MCPS and the PS reactive clones have now been characterized as to their requirements for MHC and for accessory cells. The data suggest that not only B cells, but also PS reactive T cells may contribute to the protective stimulation of neonatal responses to conjugate vaccines. One of the goals of the Children's Vaccine Initiative is to reduce the number of contacts required to immunize a child fully. To meet this goal several combined vaccines have been tested for their safety and immunogenicity in infants. We have developed a model in SW outbred mice to examine the effects of combining vaccines on the response to individual components. We initially observed that DTaP interferes with the anti-Hib response to Hib-TT Subsequent studies have confirmed the earlier findings but the interference from DTaP is not significant. We also observed that Hib-TT interfered with the response to all three types of polio, given as IPV. These studies have been repeated in inbred BALB/c mice and confirm the interference with poliotiters by Hib-TT. Preliminary studies suggest that anothe Hib conjugate, Hib-CRM197, does not cause this interference.